Protein synthesis consumes most of the cell's energy during rapid growth. As growth slows during nutrient limitation, the cell reduces protein synthesis. In order for the cell to quickly resume growth when nutrients become available, the down regulation of protein synthesis must be reversible. One method the cell employs to regulate protein synthesis is by transcriptional regulation of the protein synthesis machinery. However, this method would require de novo ribosome production when growth resumes. In contrast, relatively stable post-translational modifications like Ser/Thr phosphorylation could provide rapid and reversible control of protein synthesis by modifying existing proteins. Most bacteria contain Ser/Thr kinases and phosphatases and among the targets of these enzymes are the essential translation factors Elongation Factor Tu (EF-Tu) and Elongation Factor G (EF-G). Thus, Ser/Thr phosphorylation may regulate protein synthesis in bacteria by modulating the activities of EF-Tu and EF-G. Here, we investigate this hypothesis and identify specific Ser/Thr kinases and phosphatases that mediate the reversible phosphorylation of EF- Tu and EF-G in B. subtilis and E. coli. We will characterize how these modifications affect the function of EF-Tu and EF-G in vitro using biochemical and biophysical techniques. We also investigate the in vivo consequences of these modifications, with particular focus on sporulation in B. subtilis and stationary phase in E. coli, both physiological situations where protein synthesis is attenuated in response to nutrient limitation. This work will provide a new framework for understanding how protein synthesis is regulated in bacteria by reversible Ser/Thr phosphorylation. Our finding that Ser/Thr phosphorylation of two translation factors inhibits their activity and results in a dominan negative inhibition of elongation suggests how a process dependent on very abundant proteins can be sensitively regulated. In addition, the reversible nature of this mechanism allows cells both to enter and to exit metabolic quiescence in response to changes in nutrient availability. Finally, our characterization of these mechanisms in both E. coli and B. subtilis suggest that they are phylogenetically conserved.

Public Health Relevance

Protein synthesis is the predominant activity of growing bacteria and it consumes most of the cell's energy. Thus, when nutrients become limiting, the cells must reduce protein synthesis. Here, we describe a new mechanism of regulation of protein synthesis that occurs in both Gram-negative and Gram-positive bacteria when there are insufficient resources to permit maximal growth.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM114213-01
Application #
8862644
Study Section
Prokaryotic Cell and Molecular Biology Study Section (PCMB)
Program Officer
Bender, Michael T
Project Start
2015-04-15
Project End
2019-03-31
Budget Start
2015-04-15
Budget End
2016-03-31
Support Year
1
Fiscal Year
2015
Total Cost
$305,550
Indirect Cost
$113,050
Name
Columbia University (N.Y.)
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
621889815
City
New York
State
NY
Country
United States
Zip Code
10032
Rajagopalan, Krithika; Dworkin, Jonathan (2018) Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase in E. coli. J Bacteriol :
Libby, Elizabeth A; Dworkin, Jonathan (2017) Habits of Highly Effective Biofilms: Ion Signaling. Mol Cell 66:733-734
Sarkar, Sourav; Libby, Elizabeth A; Pidgeon, Sean E et al. (2016) In Vivo Probe of Lipid?II-Interacting Proteins. Angew Chem Int Ed Engl 55:8401-4
Sturm, Alexander; Dworkin, Jonathan (2015) Phenotypic Diversity as a Mechanism to Exit Cellular Dormancy. Curr Biol 25:2272-7
Libby, Elizabeth A; Goss, Lindsie A; Dworkin, Jonathan (2015) The Eukaryotic-Like Ser/Thr Kinase PrkC Regulates the Essential WalRK Two-Component System in Bacillus subtilis. PLoS Genet 11:e1005275
Pereira, Sandro F F; Gonzalez Jr, Ruben L; Dworkin, Jonathan (2015) Protein synthesis during cellular quiescence is inhibited by phosphorylation of a translational elongation factor. Proc Natl Acad Sci U S A 112:E3274-81